This PDF file contains the front matter associated with SPIE Proceedings Volume 6427, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

Cellular targets of photodynamic therapy include mitochondria, lysosomes, the endoplasmic reticulum (ER) and the plasma membrane. PDT can evoke necrosis, autophagy and apoptosis, or combinations of these, depending on the PDT dose, the site(s) of photodamage and the cellular phenotype. It has been established that loss of viability occurs even when the apoptotic program is inhibited. Studies assessing effects of ER or mitochondrial photodamage, involving loss of Bcl-2 function, indicate that low-dose PDT elicited a rapid autophagic response in L1210 cells. This was attributed to the ability of autophagy to recycle photodamaged organelles, and there was partial protection from loss of viability. This effect was not observed in L1210/Atg7, where autophagy was silenced. At higher PDT doses, apoptotic cells were observed within 60 min in both cell lines, but more so in L1210. The ability of L1210 cells to undergo autophagy did not offer protection from cell death at the higher PDT dose. Previous studies had indicated that autophagy can contribute to cell death, since L1210 cells that do not undergo an initial apoptotic response often contain multiple autophagic vacuoles 24 hr later. With L1210/Atg7, apoptosis alone may account for the loss of viability at an LD₉₀ PDT dose.

Pc 4, a photosensitizer first synthesized at Case Western Reserve University and now in clinical trial at University Hospitals of Cleveland, has been shown to bind preferentially and with high affinity to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components are photodamaged. In most cancer cells, apoptosis is triggered by the initial photodamage; however, in cells deficient in one of the critical intermediates of apoptosis, this process does not occur, although the cells remain as sensitive to the lethal effects of Pc 4-PDT as the apoptosis-competent cells, when cell death is determined by colony formation. Here we report that an alternative death process, autophagy, is induced in all cells tested and becomes the dominant pathway for elimination of lethally damaged cells when apoptosis is compromised. The anti-apoptotic protein Bcl-2, when overexpressed, protects only apoptosis-competent cells against loss of clonogenicity, while the autophagy inhibitor 3-methyladenine provides a markedly greater protection to apoptosis-deficient cells. The results suggest that the primary determinant of cell death is not the final pathway for elimination of the cells but the initial photodamage to critical membrane targets. In attempts to identify those targets, we have studied the role of different membrane phospholipids in the localization of Pc 4. Cardiolipin (CL) is a phospholipid found exclusively in the mitochondrial inner membrane and at the contact sites between the inner and outer membranes. Previous fluorescence resonance energy transfer studies revealed colocalization of Pc 4 and CL, which points to CL as a possible binding site and target for Pc 4. Unilamellar liposomes with different lipid compositions were used as membrane models to test the affinity of Pc 4. As revealed by the binding constants, Pc 4 does not display preferential binding to CL in these systems. Moreover, binding affinities appear to be independent of lipid composition. Localization of Pc 4 in mitochondrial membranes is likely determined by proteins or other factors not replicated in the liposomes. Studies in cells with modified CL content could report modified binding affinities.

Relatively little is known about how nitric oxide (NO) generated by tumor vascular cells or tumor cells themselves might affect the outcome of photodynamic therapy (PDT). Using a breast tumor epithelial line (COH-BR1) metabolically sensitized with protoporphyrin IX (PpIX) by pre-treating with 5-aminolevulinic acid (ALA), we have recently shown that NO from chemical donors can elicit both an immediate (NO-now) and delayed (NO-then) hyperresistance to photokilling. Cell death was mainly apoptotic when PpIX was confined to mitochondria, but mainly necrotic when it was allowed to diffuse to the cell periphery. We found that NO-now operates primarily by scavenging lipid-derived free radicals, whereas NO-then "preconditions" cells by some other mechanism. In addressing this, we have used a biologically relevant NO donor/tumor target model, viz. RAW 264.7 macrophages grown on microporous membrane inserts and COH-BR1 cells grown in culture plate wells. The RAW cells were activated with lipopolysaccharide, and 15 h later (when NO output was ~ 2 &mgr;M/h) placed over the tumor cells for 20 h, after which the latter were ALA-treated and then irradiated. Prior exposure to activated RAW macrophages reduced tumor cell photokilling by >50 %. This effect was completely lost when the RAW cells were pre-treated with the nitric oxide synthase inhibitor L-NAME, confirming that NO was involved in the hyperresistance. Results from other experiments suggest that heme oxygenase-1 and ferritin play a role in the preconditioning effect described. These studies provide new insights into how NO might modulate PDT efficacy.

Motivated by recent successes in fluorescence imaging of whole mount tissue preparations and by rapid progress in the fields of molecular imaging and molecular biology, we are exploring a number of applications of optical fluorescence imaging in superficial murine tumor models in vivo. Imaging the PDT-induced expression of the heat shock protein 70 (HSP70) in cells and in vivo is accomplished using stably transfected EMT6 cells in which the gene for GFP is under the control of the HSP70 promoter. These cells readily form solid tumors in BALB/c mice, enabling the direct imaging of the extent and time course of the activation of this promoter, with each mouse serving as its own control. Imaging of similarly transfected EMT6 cells with a HIF-1&agr;/GFP fusion protein vector enables visualization of HIF-1&agr; translocation to the nucleus. Recently, we have accomplished fluorescent labeling of surface antigens in vivo using intratumor and intravenous injection of fluorophore-conjugated antibodies. Injection of deep-red fluorophore-conjugated-anti-CD31 enables confocal fluorescence imaging of the tumor vasculature to depths of at least 100 microns. With the vessels rendered fluorescent in this way, a number of interesting studies become possible in the living mouse, including the direct visualization of photosensitizer distribution from perfused vessels. Using the appropriate fluorophore-conjugated antibodies, we have also been able to image infiltrating granulocytes in EMT6 tumors in response to PDT in vivo.

Photodynamic therapy (PDT) can be targeted toward different subcellular localizations and it is widely believed different subcellular targets vary in their sensitivity to photobiological damage. In this study, PDT-generated near-infrared singlet oxygen (¹O₂) luminescence was measured along with cell viability under two different incubation protocols: 5- aminolevulinic acid (ALA) endogenously-induced protoporphyrin IX (PpIX) and exogenous PpIX, at different incubation times. Confocal fluorescence microscopy indicated that ALA-induced PpIX (2 h) localized in the mitochondria, whereas exogenous PpIX (1 h) mainly localized to the plasma membrane. Cell viability was determined at several time points during PDT treatments using colony-forming assays, and the surviving fraction correlated well with cumulative ¹O₂ luminescence counts under both incubation protocols. Preliminary results indicate the plasma membrane is less sensitive to PDT-generated ¹O₂ than the mitochondria.

Photodynamic therapy (PDT) dose, D, is defined as the absorbed dose by the photosensitizer during photodynamic therapy. It is proportional to the product of photosensitizer concentration and the light fluence. This quantity can be directly characterized during PDT and is considered to be predictive of photodynamic efficacy under ample oxygen supply. For type-II photodynamic interaction, the cell killing is caused by the reaction of cellular receptors with singlet oxygen. The production of singlet oxygen can be expressed as &eegr;D, where &eegr; is the singlet oxygen quantum yield d is a constant under ample oxygen supply. For most PDT, it is desirable to also take into account the effect of tissue oxygenation. We have modeled the coupled kinetics equation of the concentrations of the singlet oxygen, the photosensitizers in ground and triplet states, the oxygen, and tissue receptor along with the diffusion equation governing the light transport in turbid medium. We have shown that it is possible to express eta as a function of local oxygen concentration during PDT and this expression is a good approximation to predict the production of singlet oxygen during PDT. Theoretical estimation of the correlation between the tissue oxygen concentration and hemoglobin concentration, oxygen saturation, and blood flow is presented.

Conjugates of meso-tetraphenylporphyrin to the cell targeted NLS SV40 and HIV-1 Tat 48-60 peptide sequences were synthesized on solid-phase using optimized conjugation protocols. Polar groups were introduced at the periphery of the prophyrin macrocycle, and their effect on the in vitro biological performance of the conjugates was evaluated. In vitro biological studies using the new porphyrin conjugates in human HEp2 cells showed that the conjugates bearing the HIV-1 Tat sequence were the most efficiently delivered within cells. The cellular uptake was also dependent on the nature of the substituents at the periphery of the porphyrin macrocycle. On the other hand, the conjugates containing the NLS SV40 peptide sequence and/or hydrophobic groups at the porphyrin periphery were the most phototoxic. The subcellular distribution of the conjugates depended significantly on the nature of the peptide sequence and the overall molecule charge. The conjugates delivered into the more sensitive ER were more phototoxic to the HEp2 cells than those that localized mainly in the lysosomes.

In the last three years we have prepared and studied the polar methanolic extract PMF, of the herb Hypericum perforatum L, and studied as a new, alternative photosensitizing substance for PDT. Hypericum perforatum L., as well as PMF, contains a number of naphthodianthrone derivatives (hypericins), such as hypericin and pseudohypericin, as its main photosensitizing constituents. PMF has been tested as a PDT agent in vitro in bladder cancer cells, leukemia cells, and in vivo in rat tumor bearing urinary bladder. In order to evaluate the contribution of the hypericins in the overall PDT action, and prepare a better photosensitizing extract than PMF, we have separated the extract in four main fractions (1,2,3,4), and tested their PDT effects against the HL-60 leukemic cells. The concentration of hypericins in the extracts was found 0.08% for fraction 1, 0.09% for fraction 2, 0.8% for fraction 3, and 2,8% for fraction 4. The PDT activity observed among the fractions was proportional to their hypericins concentration, thus increasing in the order of increasing number: fraction 4 > fraction 3 > fraction 2 > fraction 1. Fraction 4 proved to be the most powerful fraction. However, despite its relatively high hypericins concentration (2.8%), compared with the total extract PMF (1.37%), fraction 4 proved to be less active in the cell line tested. This result indicates that there are other photosensitizing constituents within the PMF extract which contribute significantly in the overall PDT action, and therefore the extract should be used as it is for further PDT studies, without any further purification.

The Aminolevulinic Acid (ALA) - Protoporphyrin IX (PpIX) system is unique in the world of photosensitizers in that the prodrug ALA is enzymatically transformed via the tissue of interest into fluorescently detectable levels of PpIX. This system can be used to monitor cellular metabolism of tumor tissue for applications such as therapy monitoring. Detecting PpIX fluorescence noninvasively has proven difficult due to the high levels of PpIX produced in the skin compared to other tissue both with and without ALA administration. In the current study, methods to decrease skin PpIX autofluorescence and skin PpIX fluorescence following ALA administration have been examined. Use of a purified diet is found to decrease both skin PpIX autofluorescence and skin PpIX fluorescence following ALA administration, while addition of a broad spectrum antibiotic to the water shows little effect. Following ALA administration, improved brain tumor detection is seen when skin PpIX fluorescence is photobleached via blue light prior to transmission spectroscopic measurements of tumor bearing and control animals. Both of these methods to decrease skin PpIX autofluorescence and skin PpIX fluorescence following ALA administration are shown to have a large effect on the ability to detect tumor tissue PpIX fluorescence noninvasively in vivo.

Photodynamic therapy (PDT) entails the combination of photosensitizer and light to generate cytotoxic molecules that derive from molecular oxygen (O₂). The presence of sufficient O₂ within the target tissues is critical to the efficiency of PDT. This study investigates the use of hyperbaric oxygen therapy in combination with PDT (HOTPDT) to augment the photodynamic action of methylene blue (MB) or 5-aminolevulinic acid (ALA) against gram positive and gram negative bacterial strains in vitro. Staphylococcus aureus or Pseudomonas aeruginosa were grown in trypticase soy broth as planktonic cultures (~10⁸/mL) or as established biofilms in 48 well plates (3 days old) at 32°C. Dark toxicity and PDT response in the presence or absence of HOT (2 atmospheres, 100% O₂ for 30, 60 or 120 min) was established for both MB (0-0.1 mM) and ALA (0- 1 mM) for a range of incubation times. The number of surviving colonies (CFU/mL) was plotted for each treatment groups. Light treatments (5, 10, 20 or 30 J/cm²) were conducted using an array of halogen bulbs with a red filter providing 90% transmittance over 600-800 nm at 21 mW/cm². HOT increased the dark toxicity of MB (30 min, 0.1 mM) from < 0.2 log cell kill to 0.5 log cell kill. Dark toxicity of ALA (4 hr, 1 mM) was negligible and did not increase with HOT. For non-dark toxic concentrations of MB or ALA, (0.05 mM and 1 mM respectively) HOT-PDT enhanced the antimicrobial effect of MB against Staphylococcus aureus in culture by >1 and >2 logs of cell kill (CFU/mL) at 5 and 10 J/cm² light dose respectively as compared to PDT alone. HOT-PDT also increased the anti-microbial effects of MB against Staphylococcus aureus biofilms compared to PDT, albeit less so (> 2 logs) following 10 J/cm² light dose. Anti-microbial effects of PDT using ALA were not significant for either strain with or without HOT. These data suggest that HOTPDT may be useful for improving the PDT treatment of bacterial infections.

We report the investigation of fluorescence lifetime of delta-aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX) and Photofrin© in vitro in MAT-LyLu (MLL) rat prostate adenocarcinoma cells. Photodynamic therapy (PDT) has been extensively investigated in the past decade as an effective treatment option for various types of invasive tumors. The efficacy of PDT treatment depends strongly on cell uptake and subsequent excitation of the photosensitizers. Characterization of fluorescence lifetime of these drugs provides the basis for further investigation of in vivo PDT dosage measurements using time-domain spectroscopy and imaging. Physiologically relevant concentrations of the photosensitizer solutions were prepared. A picosecond diode laser was used to excite the two drugs and the time-resolved fluorescence decay was recorded using a time-correlated single photon counting (TCSPC) system. MLL cells were incubated with the photosensitizers and were treated with light under well-oxygenated or hypoxic conditions. Fluorescence lifetime images of these cells were recorded by a confocal FLIM microscope. The measured fluorescence lifetimes of both photosensitizers are much longer than typical endogenous tissue fluorescence, which suggests time-domain methods are good candidates for in vivo PDT monitoring.

Photodynamic therapy (PDT) has gradually found its place in the treatment of malignant and non-malignant human diseases. Currently, interstitial PDT is being explored as an alternative modality for newly diagnosed and recurrent organ-confined prostate cancer. The interstitial PDT for the treatment of prostate cancer might be considered to treat prostates with permanent radioactive seeds implantation. However, the effect of implanted brachytherapy seeds on the optical fluence distribution of PDT light has not been studied before. This study investigated, for the first time, the effect of brachytherapy seed on the optical fluence distribution of 760 nm light in ex vivo models (meat and canine prostate).

Explicit dosimetry of photodynamic therapy requires detailed knowledge of the light, drug, and oxygenation distributions within the target tissue. We present a method for the optical detection and three-dimensional reconstruction of hemoglobin concentration and oxygenation and sensitizer concentration within the human prostate. Spectrally resolved diffuse transmission measurements were made using a small isotropic fiber-based white light source and an isotropic detector inserted into the prostate via parallel closed transparent catheters. The spectra were modeled using the diffusion approximation appropriate for infinite media. The optical absorption of the prostate was assumed to be a linear combination of the absorption spectra of oxy- and deoxyhemoglobin and MLu, and the scattering was assumed to be of the form A(&lgr;/&lgr;₀)^-b. The separation of absorption and scattering coefficients was accomplished based on the spectral shape of the diffuse transmission, rather than the spatial variation in intensity. By making multiple measurements at various source-detector separations, we investigate the signal-to-noise sensitivity of our algorithm. In addition, the redundancy in our source-detector position matrix creates several positions in which the tissue parameters can be reconstructed from multiple independent measurements, allowing an assessment of the repeatability of the algorithm. We find significant heterogeneity in the reconstructed optical properties; however the recovery of spectrally consistent absorption and scattering spectra is improved compared to wavelength-wise reconstruction algorithms.

Tailored light diffusers offer the flexibility of shaping the delivered light dose (fluence rate) distribution, potentially leading to conformal light delivery. Because of scattering and absorption, tissue acts as a spatial low pass filter of the diffuser's emission profile, and therefore some dose distributions with high spatial frequencies cannot be delivered. We characterize the set of attainable light dose distributions in terms of the spatial frequency of the emission profile and identify regimes where such distributions are less sensitive to changes in optical properties. Furthermore, we contrast two different algorithms to solve the inverse problem: Simulated Annealing (SA) and Non-negative Least Squares (NNLS). SA is plagued by superimposed high frequency components that do not contribute significantly to the cost. We present an iterative low pass filter that smooths the emission profile without considerably increasing the cost. A non-negative least square (NNLS) algorithm is also tested. We conclude that non-negative least squares (NNLS) is superior to simulated annealing (SA) in terms of time performance and cost minimization.

To accurately calculate light fluence rate distribution for light dosimetry in prostate photodynamic therapy (PDT), heterogeneity of optical properties has to be taken into account. Previous study has shown that a finite-element method (FEM) can be an efficient tool to deal with the optical heterogeneity. However, the calculation speed of the FEM is not suitable for real time treatment planning. In this paper, two kernel models are developed. Because the kernels are based on analytic solutions of the diffusion equation, calculations are much faster. We derived our extensions of kernel from homogeneous medium to heterogeneous medium assuming spherically symmetrical heterogeneity of optical properties. The kernel models are first developed for a point source and then extended for a linear source, which is considered a summation of point sources uniformly spaced along a line. The kernel models are compared with the FEM calculation. In application of the two kernel models to a heterogeneous prostate PDT case, both kernel models give improved light fluence rate results compared with those derived assuming homogeneous medium. In addition, kernel model 2 predicts reasonable light fluence rates and is deemed suitable for treatment planning.

Photodynamic therapy (PDT) for the treatment of prostate cancer has been demonstrated to be a safe treatment option capable of inducing tissue necrosis and decrease in prostate specific antigen (PSA). Research groups report on large variations in treatment response, possibly due to biological variations in tissue composition and short-term response to the therapeutic irradiation. Within our group, an instrument for interstitial PDT on prostate tissue that incorporates realtime treatment feedback is being developed. The treatment protocol consists of two parts. The first part incorporates the pre-treatment plan with ultrasound investigations, providing the geometry for the prostate gland and surrounding risk organs, an iterative random-search algorithm to determine near-optimal fiber positions within the reconstructed geometry and a Block-Cimmino optimization algorithm for predicting individual fiber irradiation times. During the second part, the therapeutic light delivery is combined with measurements of the light transmission signals between the optical fibers, thus monitoring the tissue effective attenuation coefficient by means of spatially resolved spectroscopy. These data are then used as input for repeated runs of the Block-Cimmino optimization algorithm. Thus, the irradiation times for individual fibers are updated throughout the treatment in order to compensate for the influence of changes in tissue composition on the light distribution at the therapeutic wavelength.

Cytokines are important messengers in cell-to-cell communications that regulate vital cellular and physiological processes, and play an important role in defining the diagnosis, prognosis and treatment response in various diseases. Although current ex vivo biochemical assays for cytokine quantitation are useful, their capabilities for studying dynamic cytokine expression in living systems are limited. Optical molecular imaging technology can help probe the spatiotemporal dynamics of cytokine expression in vivo and in real-time. We developed an in vivo optical molecular imaging strategy for monitoring one of these cytokines, the vascular endothelial growth factor (VEGF). With the imaging strategy, changes in tumoral VEGF concentration following cobalt chloride treatment and photodynamic therapy (PDT) were monitored. This was the first systematic study to test the feasibility of VEGF-targeted molecular imaging, and can potentially set the basis for online monitoring of cytokines that will help develop effective tools for diagnosis, prognosis, treatment planning and monitoring.

Photodynamic therapy (PDT) using verteporfin is widely used for treatment of age related macular degeneration (AMD). Due to non-perfect selectivity of the drug accumulation in the neovasculature some collateral damage to healthy tissue arises during the treatment. Damage to healthy structures in the eye is always a concern because of a high probability of reducing visual acuity. Two-photon (2-&ggr;) photodynamic therapy potentially offers much higher treatment selectivity than its one-photon (1-&ggr;) counterpart. By utilizing focused light for 2-&ggr; excitation, treatment volumes on the order of microliters can be achieved thus maximizing localized insult to abnormal blood vessels and sparing healthy tissue. We propose that 2-&ggr; photodynamic therapy will be valuable in the treatment of choroidal neovascularization secondary to age related macular degeneration as well as other conditions. To ascertain feasibility of 2-&ggr; photodynamic therapy we measured 2-&ggr; spectrum and cross sections of verteporfin (80 GM at 940 nm, 1 GM = 10^-50 cm⁴s/photon), chlorin e6 (14 GM at 800 nm) and tetrasulfonated aluminum phthalocyanine (140 GM at 900 nm) and investigated their in vitro efficiency under 2-&ggr; excitation. Only verteporfin demonstrated cell kill under the used irradiation parameters (average light intensity 9.1 mW, wavelength 850 nm, total light dose 6900 J/cm²). Dorsal skinfold window chamber model in mouse was used to test efficiency of 2-&ggr; PDT with verteporfin in vivo. Although we were able to induce photodynamic damage to a blood vessel using 1-&ggr; excitation, 2-&ggr; excitation resulted in no visible damage to irradiated blood vessel. The most probable reason is low efficiency of verteporfin as a 2-&ggr; photosensitizer. We also report 2-&ggr; spectrum of new photosensitizer, HCC4 (4300 GM at 830 nm), specifically designed for efficient 2-&ggr; excitation.

The parameters which limit supply of photosensitizer to the cancer cells in a solid tumor were systematically analyzed using microvascular transport modeling and histology data from frozen sections. In particular the vascular permeability transport coefficient and the effective interstitial diffusion coefficient were quantified for verteporfin-for-injection delivery of benzoporphyrin derivative (BPD). Orthotopic tumors had a higher permeability and diffusion coefficients (P_d= 0.036 &mgr;m/s and D = 1.6 &mgr;m²/s, respectively) as compared to subcutaneously grown tumors (P_d = 0.025 &mgr;m/s and D = 0.9 &mgr;m²/s, respectively), likely due to the fact that the vessel patterns are more homogeneous orthotopically. In general, large inter-subject and intra-tumor variability exist in the verteporfin concentration, in the range of 25% in plasma concentration and in the range of 20% for tissue concentrations, predominantly due to these micro-regional variations in transport. However, the average individual uptake of photosensitizer in tumor tissue was only correlated to the total vascular area within the tumor (R2 = 64.1%, p < 0.001). The data is consistent with a view that micro-regional variation in the vascular permeability, interstitial diffusion rate, all contributes the spatial heterogeneity observed in verteporfin uptake, but that average supply to the tissue is limited by the total area of perfused blood vessels. This study presents a method to systematically analyze microheterogeneity as well as possible methods to increase delivery and homogeneity of photosensitizer within tumor tissue.

Meso-tetra(hydroxyphenyl)chlorin (mTHPC)(international generic name Temoporfin) is a potent photosensitizer used for photodynamic therapy (PDT). In this study the pharmacokinetics of a systemically administered novel lipid formulation of Temoporfin in a murine tumor model has been investigated. Fluorescence spectroscopy measurements were performed at several time intervals following drug administration, yielding information on the Temoporfin concentration within excised internal organs as a function of time after injection. Both point-monitoring and imaging setups were used. The acquired fluorescence data were correlated to the concentration of Temoporfin obtained with High Performance Liquid Chromatography (HPLC). There was a significant correlation between the fluorescence methods and HPLC for most organs investigated. The pharmacokinetics of this new liposomal formulation of Temoporfin exhibited a rather flat temporal profile in the time interval 2-8 hours in this study.

This work is devoted to investigation of several new photosensitizers on a base of bacteriochlorin p macrocycle. Investigations of photosensitizers in vivo and experimental PDT were performed on F₁ mice bearing Erlich tumor and BDF₁ mice bearing B16 melanoma. Spectra of fluorescence and absorption of tissue were studied in vivo using fiberoptic spectroanalyzer LESA. All investigated photosensitizers possess intensive absorption in a range of 730-800 nm, where the intrinsic absorption of the biological tissue has its minimum. Dynamics and selectivity of sensitizer accumulation in tumor and normal tissue were estimated from spectra of absorption or fluorescence of sensitized tissue in vivo. The investigation has shown that the optimum time range to start PDT irradiation of tumor is 10-30 min after administration. All photosensitizers clear from the normal tissue in less than 24 hours. Photodynamic efficiency was estimated by comparison of the tumor volume growth in control and treated animal groups. All studied photosensitizers have shown high photodynamic efficiency of relatively large tumors, index of tumor growth inhibition exceeded 70%. Bacteriochlorin p N-methoxycycloimide oxyme methyl ester was also shown to be efficient for PDT of B16 melanoma, causing tumor growth inhibition more than 90%.

Current work is devoted to investigation of tetra-3-phenylthio-tetra-5-t-butylphthalocyanine [(PhS)₄(t-Bu)₄PcH₂], aluminium hydroxyde tetra-3-phenylthiophthalocyanine [(PhS)₄PcAlOH] and zinc tetra-3-phenylthiophthalocyanine [(PhS)₄PcZn] as potential photosensitizers of near-infrared range. Investigations were performed on F₁ mice bearing Erlich tumor. Photosensitizers were administered intravenously in liposomal form at doses of 4-10 mg/kg. Dynamic and selectivity of sensitizers' accumulation in tumor were estimated in vivo from fluorescence and absorption spectra of sensitized tissue. Photosensitizers have shown high selectivity of accumulation in tumor comparing to normal tissue of mice. Maxima of selectivity for (PhS)₄(t-Bu)₄PcH₂, (PhS)₄PcZn and (PhS)₄PcAlOH achieve the values up to 2.5:1, 5:1 and 8:1 respectively. All photosensitizers completely clear from the normal tissue in 7-8 days. For PDT investigations tumors were irradiated using 732 nm laser with power density of 100-500 mW/cm² and light dose density up to 400 J/cm². The photodynamic efficiency was estimated using the parameter of tumor growth inhibition (TGI). All photosensitizers had shown high photodynamic efficiency of relatively large tumors. PDT using (PhS)₄PcAlOH and (PhS)₄(t-Bu)₄PcH₂ caused pronounced TGI exceeding 80%. Using (PhS)₄PcZn caused moderate TGI of 60%. Investigations have shown that liposomal forms of phenylthiosubstituted phthalocyanine derivatives may be used to develop new efficient photosensitizers for PDT.

Fluorescence technique appears very important for the diagnosis of cancer. Fluorescence detection has advantages over other light-based investigation methods: high sensitivity, high speed, and safety. Renal cell carcinoma (RCC) accounts for approximately 3% of new cancer incidence and mortality in the United States. Unfortunately many RCC masses remain asymptomatic and nonpalpable until they are advanced. Diagnosis and localization of early carcinoma play an important role in the prevention and curative treatment of RCC. Certain drugs or chemicals such as porphyrin derivatives accumulate substantially more in tumors than normal tissues. The autofluorescence of blood porphyrin of healthy and tumor induced male SCID mice was analyzed using fluorescence and excitation spectroscopy. A significant contrast between normal and tumor blood could be established. Blood porphyrin fluorophore showed enhanced fluorescence band (around 630 nm) in function of the tumor growth. This indicates that either the autofluorescence intensity of the blood fluorescence may provide a good parameter for the "first approximation" characterization of the tumor stage.

Effective photodynamic therapy depends on an adequate supply of photosensitizer, oxygen, and light fluence. During light exposure, the rapid depletion of molecular oxygen within tissue can lead to the development of hypoxic regions, which decreases the generation of reactive oxygen species and can result in non-uniform tissue necrosis. The Optical Pharmacokinetic System (OPS) is a fiber-optic based spectroscopy device that may be able to monitor local tissue oxygen concentrations during treatment and identify problematic hypoxic regions in vivo. However, the 'bulk' signal detected by the OPS is potentially limited in its ability to discern the development of small hypoxic regions within tissue. This study employs a Monte Carlo simulation of the elastically-scattered light as measured by the OPS to investigate the effect of heterogeneous chromophore distributions on the detected signal. The model tissue geometry is constructed to mimic tissue in vivo, with discrete capillaries interspersed throughout. Tissue optical properties are specified spatially, allowing investigation of heterogeneous chromophore distribution. Simulations investigate the effect that discrete, highly absorbing regions within a measured sample have on the light collected by the OPS. Simulations also consider OPS measurement of a sample with a depth-dependent chromophore concentration gradient and quantitate the ability of the OPS to detect the presence of a sub-population of hypoxic vessels within a network of oxygenated vessels.

Esophageal photodynamic therapy (PDT) is performed using a photosensitizing agent activated by light delivered via a cylindrically symmetric delivery device containing a diffusing optical fiber. In PDT treatment of dysplastic Barrett's esophagus, considerable variability in results is observed due to the non-uniform delivery of treatment light caused by source geometry and by luminal collapse. We compare the fluence rate at the tissue surface resulting from illumination with bare fiber, a centering balloon catheter (X-Cell, Cooke, Inc), and a fixed diameter transparent dilating catheter (Optical Dilator, Inscope, Ethicon ES). Measurements were made in a solid esophagus-simulating phantom illuminated by 2.5 and 5 cm diffusing fibers with and without each delivery device. The diffuser was coupled to a 630 nm dye laser pumped by a 532 nm KTP laser (LaserScope, Inc.) The total power emitted by the diffuser was 1W. The fluence rate as a function of position along the cavity was measured by a calibrated photodiode connected to an optical fiber with a 0.5 mm isotropic scattering tip, which was moved by a computer-controlled positioner. The mean fluence rate measured when the phantom was illuminated using either the centering balloon or the rigid dilator was approximately 50% less than that measured with a bare fiber. The decrease in fluence rate is due to attenuation of the primary light beam and to reduction in scattering from laterally adjacent points in the phantom. The importance of each of these effects as a function of tissue optical properties was confirmed using Monte Carlo simulation.

Photodynamic therapy (PDT) is an approved treatment modality for Barrett's and invasive esophageal carcinoma. Proper Combination of photosentizing agent, oxygen, and a specific wavelength of light to activate the photosentizing agents is necessary for the cytotoxic destruction of cancerous cells by PDT. As a light source expensive solid-state laser sources currently are being used for the treatment. Inexpensive semiconductor lasers have been suggested for the light delivery system, however packaging of semiconductor lasers for optimal optical power output is challenging. In this paper, we present a multidirectional direct water-cooling of semiconductor lasers that provides a better efficiency than the conventional unidirectional cooling. AlGaAsP lasers were tested under de-ionized (DI) water and it is shown that the optical power output of the lasers under the DI water is much higher than that of the uni-directional cooling of lasers. Also, in this paper we discuss how direct DI water-cooling can optimize power output of semiconductor lasers. Thereafter an optimal design of the semiconductor laser package is shown with the DI water-cooling system. Further, a microwave antenna is designed which is to be imprinted on to a balloon catheter in order to provide local heating of esophagus, leading to an increase in local oxygenation of the tumor to generate an effective level of singlet oxygen for cellular death. Finally the optimal level of light energy that is required to achieve the expected level of singlet oxygen is modeled to design an efficient PDT protocol.

Biomedical applications of near-infrared Raman spectroscopy have increased their importance at the last ten years. This technique can determinate the molecular composition of materials, allowing a sensible and fast biological diagnosis. It has showed to be a promising tool for health diagnosis due to its high sensibility. Colorectal cancer (CRC) is one of the most common malignant tumors in humans beings. In the last decades many experimental models have been developed in animals based in the use of chemical composites to induce the formation and development of these tumors, many of them present similar characteristics to those of natural occurrence aiming to the attainment of information on genesis, evolution, as well as diagnosis and more efficient therapies for treating these neoplasias. Amongst the most used chemical composites is the 1,2- dimetilhydrazine (DMH) because its morphological and histological similarity to those tumors. This study aims to compare in vivo normal colon tissue and tumoral colon tissue, induced by DMH, in rats by near-infrared Raman spectroscopy to permit the use in the near future for an efficient diagnosis in real time besides being useful as an auxiliary method for several therapies, including the photodynamic therapy.

In order to investigate the interaction between the triplet state T₁ and ground state oxygen ³O₂ during pulsed excitation photodynamic therapy (PDT), we measured the phosphorescence and singlet oxygen ¹O₂ fluorescence time-resolved waveform. The phosphorescence time-resolved waveform from clinical photosensitizers has not been obtained because this signal was buried in the photosensitizer fluorescence. We constructed the experimental setup with a spectral and temporal filter to select the phosphorescence signals from the Photofrin(II)^(R) and Talaporfin sodium solution. The lifetimes and spectrums of the measured luminescence coincided with the phosphorescence characteristics, respectively. We obtained the phosphorescence time-resolved waveforms from the clinical photosensitizer solutions successfully. The ¹O₂ fluorescence time-resolved waveforms from these photosensitizers were measured with an IR-PMT with a photon counter. The fluorescence time-resolved waveforms of each photosensitizer were also obtained by the authors. We could consequently describe sequential generation of three time-resolved waveforms throughout the photosensitive reaction in the clinical photosensitizers. We think we may evaluate the photoseisitizer characteristics by these waveforms.

Introduction: Photodynamic therapy (PDT) is a treatment for cancer requiring activation of a photosensitizer for light-mediated tumor cytotoxicity. PDT is limited by tissue penetration because visible light is required for photosensitizer activation. Sunstones^TM are rare-earth phosphors which up-convert energy from infrared wavelengths to emit higher energy in the visible spectrum. We utilized this unique characteristic to generate light of appropriate intensity and wavelength for photosensitizer activation and subsequent tumor cell eradication. Methods: Sunstones^TM with infrared absorption at 808 and 980nm and visible two-photon emission at 549.9 and 663.1nm were used. A murine NSCLC line was used to determine in vitro toxicity of Sunstones^TM and dose response curves for Sunstones^TM-mediated PDT. Human NSCLC cells were incubated with/without Photofrin^TM (photosensitizer). Experimental groups included: Infrared light treatment (IR), IR+Photofrin^TM, IR+Sunstones^TM, and IR+Sunstones^TM+Photofrin^TM. Groups were exposed to 2.5W of 808nm light and assayed for metabolic activity. Results: In vitro toxicity assays showed no significant toxicological side effects after 1 week incubation with Sunstones^TM and demonstrated linear response in cytotoxicity as treatment times and infrared dose increased. IR+Sunstones^TM+Photofri^TM group showed significantly decreased metabolic activity compared to control cells, cells treated with IR+Sunstones^TM, and IR alone. Conclusion: Sunstones^TM are nontoxic nanocrystals capable of activating photosensitizers for PDT. Future directions include conjugation of up-converters to a novel photosensitizer and managing the selective uptake of conjugate by tumor cells.

Photodynamic therapy (PDT) is a treatment modality which has been shown to be effective for both malignant and non-malignant diseases. New photosensitizers such as hexyl-aminolevulinate (HAL) may increase the efficiency of PDT. HAL penetrates into the cell where the photosensitizer protoporphyrin IX (PPIX) is produced endogenously. In a previous study on HAL based PDT treatment of rat bladder cancer (AY-27 transitional cell carcinoma), a depression of the optical reflectance spectra after treatment was observed in some of the animals. This depression of the spectra was caused by metHemoglobin (metHb). MetHb is an indication of oxidative stress, and can be formed as a result of for instance UV-radiation and heating of blood. The aim of this study was to identify if metHb can be formed in vitro as a result of oxidative stress caused by singlet oxygen and ROS produced during PDT. Methemoglobin formed during PDT might thus be used as an indirect measure of the photochemical processes. This may help predict the PDT treatment outcome. Red blood cells mixed with AY-27 cells exposed to HAL, or PPIX received light treatment, and the changes in the absorption spectra were measured spectrophotometrically. The methemoglobin absorbance spectrum was also studied, and found to be strongly dependant on pH. Hemolysis of erythrocytes by PDT was found, however no metHb was formed in vitro.

A new anionic porphyrin-phosphate conjugate has been made as the substrate of phosphatase to evaluate its cellular-uptake and potential targeting on cancer cells, taking advantage of the over-expression of phosphatases associated with the development of cancers. The phosphate groups increase the hydrophilicity of porphyrin dityrosine phosphate and facilitate its formulation in aqueous solvent. Upon hydrolysis by phosphatase after cellular uptaking, the more hydrophobic porphyrin-dityrosine promises to give better cellular retention. Indeed, the phosphate conjugate displayed a much better PDT effect than that of the parent porphyrin at the same concentration (10 &mgr;M) and light dosage on HeLa cells, indicating the enzyme-cleavage reaction occurred in HeLa cells plays a role. Photosenzitizers utilizing enzyme-cleavage might be a promising approach for photodynamic therapy.

Multi-sensory fiber-optic probes using nanocrystals as the sensing materials are shown to overcome most limitations imposed by those using organic dyes as the fluence-rate sensing materials. These nanocrystals are shown to be excitable by a wide range of wavelengths covering the entire tissue transparent window utilized by various light-enabled treatment modalities. The optical response of the sensors made from the nanocrystals is shown to be linear without any sign of photobleaching and sensor crosstalk over a wide range of irradiance and fluence. Fiber probes using the nanocrystals are promising in offering high spatial resolution in fluence-rate dosimetry for photodynamic therapy and photothermal treatment.

A six-year old Holstein cow with an eye cancer (ocular squamous cell carcinoma) involving the third eyelid and conjunctiva was submitted to photodynamic therapy using intratumoral 20% aminolevulinic acid (5-ALA - Aldrich Chemical Company, Milwaukee, USA) and a light emitting diode (LED - VET LED - MMOptics^(R)) with wavelength between 600 and 700 nm, 2 cm diameter circular light beam, power of 150 mW, light dose of 50 J/cm² as a source of irradiation. Fifteen days after the experimental procedure we observed about 50% tumor reduction and complete remission after 3 months. Relapse was not observed up to 12 months after the treatment. Although the study only includes one animal not allowing definite conclusions, it indicates that PDT represents a safe and technically feasible approach in the treatment of eye cancer in cattle.

Objective To study the effects of HMME-based photodynamic therapy on proliferation and apoptosis of rabbit vascular smooth muscle cells(VSMCs). Method The cytotoxic effect of HMME-PDT on rabbit vascular smooth muscle cells was studied by means of Trypan Blue assay, HMME at 10&mgr;g/ml concentration and the light dose at 2.4~4.8 J/cm² were selected in the studies. The morphological character 24h post-PDT was investigated by HE Staining. Annexin V and propidium iodide (PI) binding assays were performed to analyze the characteristics of cell death after HMME-PDT. Furthermore, The intracellular distributions of the HMME were measured by the confocal laser scanning microscope. Result It was showed the photocytotoxity to VSMC cells was dose related by Trypan Blue assay. Histology observing suggests HMME-PDT could induce cell death through apoptosis or necrosis, and the apoptosic rate was up to 50.5% by AnnexinV /PI assay. Moreover, the fluorescence images of HMME intracellular localization demonstrated that the HMME diffused into the mitochondria. Conclusion HMME-PDT could significantly inhibite VSMC proliferation and induce apoptosis.

Introduction and background: We have reported on the polar methanolic fraction (PMF) of Hypericum Perforatum L as a novel photosensitizing agent for photodynamic therapy (PDT) and photodynamic diagnosis (PDD). PMF has been tested in human leukemic cells, HL-60 cells, cord blood hemopoietic progenitor cells, bladder cancers derived from metastatic lymph node (T-24) and primary papillary bladder lesion (RT-4). However, the mechanisms of the effects of PMF on these human cell lines have not been elucidated. We have investigated mechanisms of PMF + light versus PMF-alone (dark experiment) in T-24 human bladder cancer cells. Methods: PMF was prepared from an aerial herb of HPL which was brewed in methanol and extracted with ether and methanol. Stock solutions of PMF were made in DSMO and stored in dark conditions. PMF contains 0.57% hypericin and 2.52% hyperforin. The T24 cell line was obtained from American Type Culture Collection (ATCC). In PDT treatment, PMF (60&mgr;g/ml) was incubated with cells, which were excited with laser light (630nm) 24 hours later. Apoptosis was determined by DNA fragmentation/laddering assay. DNA isolation was performed according to the manufacture's instructions with the Kit (Oncogene Kit#AM41). Isolated DNA samples were separated by electrophoresis in 1.5% in agarose gels and bands were visualized by ethidium bromide labeling. The initial cell cycle analysis and phase distribution was by flow cytometry. DNA synthesis was measured by [3H] thymidine incorporation, and cell cycle regulatory proteins were assayed by Western immunoblot. Results: The results of the flow cytometry showed PMF +light induced significant (40%) apoptosis in T24 cells, whereas Light or PMF alone produced little apoptosis. The percentage of cells in G₀/G₁ phase was decreased by 25% and in G2/M phase by 38%. The main impact was observed on the S phase which was blocked by 78% from the specific photocytotoxic process. DNA laddering analysis showed that PMF (60&mgr;g/ml) + light at 630nm induced DNA fragmentation in a light dose-dependent manner; in contrast, PMF or light alone did not induce DNA fragmentation. In separate experiments, PMF alone treatment produced a dose-dependent DNA synthesis with a 90% inhibition at a concentration of 25&mgr;g/ml (IC90 = 25&mgr;g/ml). Expression of p53 and p27 cell cycle regulatory proteins was not altered by PMF alone, however, a dose-dependent increase in p21 expression was observed that correlates with PMF concentrations. Cyclin A and cyclin B protein levels showed a clear decrease inverse to the concentration of PMF. In the absence of light treatment, flow cytometry analysis showed that PMF alone results in G₀/G₁ cell cycle arrest, with a 2-fold increase in G₀/G₁ cells concomitant with 50% decrease in cells in both S and G₂/M phases. However, flow cytometry on PMF alone-treated cells did not show sub G₀/G₁ peak, further evidence of the lack of apoptosis as a mechanism of effect of PMF in the dark. Conclusions: With respect to light treatment, apoptosis appears to play a vital role in PDT-induced cytotoxicity. The flow cytometry and DNA laddering results revealed that T24 cells demonstrated apoptotic responses in PMF-mediated PDT. Experiments conducted with PMF alone showed a dose-dependent inhibition of DNA synthesis associated with G₀/G₁ cell cycle arrest and the extract is able to coordinate changes in key cell cycle regulatory proteins in human bladder cancer cells. Both experimental conditions suggest PMF as a potent and effect anti-proliferative agent in cancer chemoprevention and therapy of human urothelial carcinoma cells.